Detection and preliminary characterization of two enzymes involved in biosynthesis of platelet-activating factor in mouse oocytes, zygotes and preimplantation embryos: dithiothreitol-insensitive cytidinediphosphocholine: 1-O-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase and acetyl-coenzyme A:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase

Abstract

The aim of this study was to determine whether the final enzymes in the two biosynthetic pathways for platelet-activating factor (PAF) (the 'de novo' and the 'membrane remodelling' pathways) are present in mouse embryos, zygotes and oocytes. The enzymes are dithiothreitol-insensitive cytidinediphosphocholine: 1-O-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (cholinephosphotransferase) in the de novo pathway and acetyl-coenzyme A:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase (acetyltransferase) in the membrane remodelling pathway. Activity of both enzymes was detected in the unfertilized oocyte, the zygote and also in the preimplantation embryo (48, 72 and 96 h after the ovulatory injection of hCG). In both cases the activity was destroyed by boiling and increased linearly with incubation time and the concentration of embryo homogenate present, indicating that the reactions were catalysed by enzymes. The product of the reactions was confirmed as PAF using HPLC and structural analyses by enzymatic digestion. Cholinephosphotransferase required Mg2+ and was inhibited by Ca2+, while acetyltransferase required the presence of NaF (a phosphatase inhibitor). The activity of cholinephosphotransferase was similar in unfertilized oocytes and zygotes, and did not change significantly with advancing developmental stage in preimplantation embryos. Acetyltransferase had a significantly lower specific activity (0.078 +/- 0.044 fmol PAF per oocyte per min, mean +/- SEM) in unfertilized oocytes than in zygotes of corresponding age (0.358 +/- 0.097 fmol PAF per zygote per min) (P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)